Nucleic acid amplification techniques have provided powerful tools for detection and analysis of small amounts of nucleic acids. The extreme sensitivity of such methods has lead to attempts to develop them for early diagnosis of infectious and genetic diseases, isolation of genes for analysis, and detection of specific nucleic acids in forensic medicine. Nucleic acid amplification techniques can be grouped according to the temperature requirements of the procedure. The polymerase chain reaction (PCR), ligase chain reaction (LCR) and transcription-based amplification require repeated cycling of the reaction between high (85.degree. C.-100.degree. C.) and low (30.degree. C.-40.degree. C.) temperatures to regenerate single stranded target molecules for amplification. In contrast, methods such as Strand Displacement Amplification (SDA), self-sustained sequence replication (3SR) and the Q.beta. replicase system are isothermal reactions which can be performed at a constant low temperature (usually about 30.degree.-40.degree. C.).
One of the best-known nucleic acid amplification methods is the Polymerase Chain Reaction (PCR). This method is described by R. K. Saiki, et al. (1985. Science 230, 1350-1354) and in U.S. Pat. No. 4,683,195, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,800,159. Briefly, to amplify a target sequence using the PCR, two primers complementary to sequences flanking the target sequence are hybridized (one to each of the opposite complementary strands) and extended, using the target sequence as a template, by addition of deoxyribonucleotides and a DNA polymerase. After extension, the temperature of the reaction is raised to separate the newly-synthesized strand from the template, then lowered to reanneal the primers and repeat the extension process. Due to the characteristic cycling of the reaction temperature, the PCR requires the use of a heat stable polymerase such as Taq polymerase.
In contrast, Strand Displacement Amplification (SDA) is an isothermal method of nucleic acid amplification in which extension of primers, displacement of single stranded extension products, annealing of primers to the extension products (or the original target sequence) and subsequent extension of the primers occurs concurrently in the reaction mix. This is in contrast to the PCR, in which the steps of the reaction occur in discrete phases or cycles as a result of the temperature constraints of the reaction. SDA is based upon 1) the ability of a restriction endonuclease to nick the unmodified strand of a hemiphosphorothioate form of its double stranded recognition site and 2) the ability of certain polymerases to initiate replication at the nick and displace the downstream non-template strand. After an initial incubation at increased temperature (about 95.degree. C.) to denature double stranded target sequences for annealing of the primers, subsequent polymerization and displacement of newly synthesized strands takes place at a constant temperature (usually about 37.degree. C.). Production of each new copy of the target sequence consists of five steps: 1) binding of amplification primers to an original target sequence or a displaced single-stranded extension product previously polymerized, 2) extension of the primers by exonuclease deficient (exo.sup.-) klenow polymerase incorporating an .alpha.-thio deoxynucleoside triphosphate, 3) nicking of a hemiphosphorothioate double stranded restriction site, 4) dissociation of the restriction enzyme from the nick site, and 5) extension from the 3' end of the nick by exo.sup.- klenow with displacement of the downstream non-template strand. Nicking, polymerization and displacement occur concurrently and continuously at a constant temperature because extension from the nick regenerates another nickable restriction site. When primers which hybridize to both strands of a double stranded target sequence are used, amplification is exponential, as the sense and antisense strands serve as templates for the opposite primer in subsequent rounds of amplification. SDA is described by G. T. Walker, et al. (1992a. Proc. Natl. Acad. Sci. USA 89, 392-396 and 1992b. Nuc. Acids. Res. 20, 1691-1696). Examples of restriction enzymes which nick their double stranded recognition sites when an .alpha.-thio dNTP is incorporated are HincII, HindII, AvaI, NciI and Fnu4HI. All of these restriction enzymes and others which display the required nicking activity are suitable for use in SDA. The Walker, et at. disclosures are hereby incorporated by reference and details of the SDA method are found in the following Examples.
Targets for amplification by SDA may be prepared by fragmenting larger nucleic acids by restriction with the endonuclease used in the SDA reaction (e.g., HincII). However, for in situ amplification it is most preferred that target nucleic acids having the selected restriction endonuclease recognition sites for nicking in the SDA reaction be generated as described by Walker, et al. (1992b, supra). This target generation scheme is also described in U.S. Pat. No. 5,270,184, the disclosure of which is hereby incorporated by reference. This method for generation of SDA-amplifiable target sequences comprises heat denaturing double stranded nucleic acids containing the target sequence and hybridizing four primers to the target sequence. Two of the primers (S.sub.1 and S.sub.2) are SDA amplification primers as defined below, with target binding sequences near their 3' ends and restriction enzyme recognition sites 5' to the target binding sequences. When both amplification primers are used amplification is exponential, however, use of only one amplification primer results in linear amplification of the target sequence. The other two primers (B.sub.1 and B.sub.2) are external primers as defined below and consist only of target binding sequences. S.sub.1 and S.sub.2 bind to opposite strands of double stranded nucleic acids flanking the target sequence. B.sub.1 and B.sub.2 bind to the target sequence 5' (i.e., upstream) of S.sub.1 and S.sub.2, respectively. Exonuclease deficient klenow polymerase (exo.sup.- klenow polymerase) is then used to simultaneously extend all four primers in the presence of three deoxynucleoside triphosphates and one modified deoxynucleoside triphosphate (e.g., deoxyadenosine 5'-[.alpha.-thio]triphosphate dATP[.alpha.S]). Extension of S.sub.1 and S.sub.2 produces two extension products, S.sub.1 -ext and S.sub.2 -ext. Extension of B.sub.1 and B.sub.2 results in displacement of the downstream S.sub.1 and S.sub.2 extension products from the original target sequence template. The displaced, single stranded S.sub.1 extension product serves as a target for binding of S.sub.2 and B.sub.2. Similarly, the displaced, single stranded S.sub.2 extension product serves as a target for binding of S.sub.1 and B.sub.1. All four primers are then extended on the S.sub.1 -ext and S.sub.2 -ext templates to produce a second pair of extension products which are displaced by extension of the external primers as before. Binding and extension of complementary amplification primers on these displaced extension products results in synthesis of a complementary strand. This produces two double stranded nucleic acid fragments with hemimodified restriction enzyme recognition sites at each end which are suitable for amplification by SDA. The extended external primers hybridized to S.sub.1 -ext and S.sub.2 -ext form two larger double stranded fragments with hemimodified restriction enzyme recognition sites at only one end. As in SDA, the individual steps of the target generation reaction occur concurrently and continuously, generating target sequences with the required recognition sequences at the ends for nicking by the restriction enzyme in SDA. As all of the components of the SDA reaction are already present in the target generation reaction, target sequences generated automatically and continuously enter the SDA cycle and are amplified.
In situ methods of nucleic acid analysis allow detection and localization of specific nucleic acid sequences within morphologically intact cells. In situ methods of nucleic acid analysis have conventionally been accomplished by direct hybridization of labeled probes, for example as described in U.S. Pat. No. 4,888,278. However, such direct hybridization methods, while specific for the nucleic acid of interest, may not be sufficiently sensitive to detect very low copy numbers of the nucleic acid in all cases. As a means for detecting very low copy numbers, in situ amplification of the target sequence prior to in situ detection has been of great interest. In situ nucleic acid amplification methods have the potential to be more sensitive that conventional solution amplification because the cell may concentrate the amplification product, thereby allowing detection of fewer molecules than is possible when amplification products are free to diffuse or are diluted by the contents of cells which do not contain the sequence of interest. Because the nucleic acid is not extracted from the cell prior to the analysis, in situ methods provide information as to which cells in a population contain a particular nucleic acid and further permit analysis of the nucleic acid in the context of the biochemical and morphological characteristics of the cell. Prior to the present invention, in situ amplification methods have only been developed for the PCR (O. Bagasra and R. Pomerantz. 1993. AIDS Research and Human Retroviruses 9(1), 69-76; G. Nuovo, et al. 1992. Diag. Molec. Pathol. 1(2), 98-102; M. J. Embleton, et al. 1992. Nuc. Acids Res. 20(15), 3831-3837; J. Embretson, et al. 1993. Proc. Natl. Acad. Sci. USA 90, 357-361; P. Komminoth, et al. 1992. Diag. Molec. Pathol. 1(2), 85-97; K. P. Chile, et al. 1992. J. Histochem. Cytochem. 40(3), 333-341; Haase, et al. 1990. Proc. Natl. Acad. Sci. USA 87, 971-4975; O. Bagasra, et al. 1992. New Engl. J. Med. 326(21), 1385-1391; Patterson, et al. 1993. Science 260, 976-979). However, the multiple cycles of heating and stringent hybridization conditions required by the PCR to achieve its sensitivity are not well tolerated by tissues and cells. Diffusion of the amplified sequences out of the cells is increased by the repeated heating, resulting in increased diffuse signal throughout the sample. To attempt to reduce the loss of PCR products from the cell, long fixation times (15 hours to days) with cross-linking fixatives are considered essential for successful in situ amplification by the PCR. As a result of the extensive fixation, it is also considered critical to treat the fixed cells with protease prior to amplification (G. Nuovo, et al. 1992. Diag. Molec. Pathol. 1(2), 98-102).
The ability of SDA to amplify low copy-number target sequences at 37.degree. C. made it desirable to attempt to develop methods for in situ nucleic acid amplification using this technique. However, the many significant differences between PCR and SDA amplification reaction protocols made it highly uncertain whether or not in situ amplification could be successfully performed at a constant, relatively low temperature to minimize destructive effects on the cellular morphology. Both the in situ PCR and in situ SDA employ fixing of the cells to maintain cell integrity. At the lower temperatures of SDA, nucleic acids within the fixed cell are therefore more stably crosslinked with proteins which may physically interfere with hybridization and prevent access of polymerase, probes and/or restriction enzymes to the target sequence. Access of polymerase and/or restriction enzymes to the probes or amplicons may also be inhibited by associated proteins, thus preventing amplification. In the PCR, it is likely that heating assists in partially or wholly reversing the fixation or the effects of fixation, releasing the associated proteins from the nucleic acids or decreasing their affinity and freeing them for amplification. No such release of interfering proteins would be expected in isothermal amplification reactions. In addition, the heating steps for the PCR may repetitively denature inter- and intra-strand hybridization allowing the primers better access to the target. No such repetitive heating occurs with SDA. Further, the phosphorothioates used in SDA may form disulfide bonds with cellular proteins, preventing access of reagents to the target.
Further, the amplification products of the PCR are generally larger in size than those produced by SDA. SDA amplification products would therefore be even more likely than PCR amplification products to diffuse out of the cell. It was not previously known whether or not the amplification products of SDA would be large enough to be retained by the cell after in situ amplification. Although in situ PCR amplification has been performed on formaldehyde-fixed cells, it was uncertain what affect fixing and the formaldehyde fixative itself would have on the particular enzymatic reactions required by SDA--e.g., nicking of a hemiphosphorothioate recognition site by a restriction enzyme and the displacing activity of exo.sup.- klenow polymerase. Not only does the crosslinking which occurs in the fixing process potentially and unpredictably exclude certain molecules from the interior of the cell, formaldehyde is known to interact with nucleic acids and may inhibit restriction by endonucleases and other enzymatic activities.
For purposes of the instant disclosure, the following terms are defined as follows:
An amplification primer is a primer for amplification of a target sequence by hybridization and extension of the primer. For SDA, the 3' end of the amplification primer (the target binding sequence) hybridizes at the 3' end of the target sequence and comprises a recognition site for a restriction enzyme near its 5' end. The target binding sequence is generally approximately 10-20 base pairs in length. The restriction enzyme recognition site is a nucleotide sequence recognized by a restriction enzyme which will nick one strand of a DNA duplex when the recognition site is hemimodified, as described by Walker, et al. (1992a), supra. A hemimodified recognition site is a double stranded recognition site for a restriction enzyme in which one strand contains at least one derivatized nucleotide which prevents cutting of that strand by the restriction enzyme. The other strand of the hemimodified recognition site does not contain derivatized nucleotides and is nicked by the restriction enzyme. Any hemimodified restriction enzyme recognition site which is nickable by a restriction enzyme is suitable for use in SDA. Examples of the preferred hemimodified recognition sites are hemiphosphorothioated recognition sites for the restriction enzymes HincII, HindII, AvaI, NciI and Fnu4HI. Amplification primers for SDA are designated S.sub.1 and S.sub.2 by Walker, et al. (1992b), supra.
A "bumper" or external primer is a primer which anneals to a target sequence upstream of an amplification primer, such that extension of the external primer displaces the downstream primer and its extension product. The bumper primers therefore consist only of target binding sequences and are designed so that they anneal to the target sequence close enough to the amplification primers to displace them when extended. External primers are designated B.sub.1 and B.sub.2 by Walker, et al. (1992b), supra. Extension of external primers is one method for displacing the extension products of amplification primers, but heating may also be suitable in certain cases.
The terms target or target sequence refer to nucleic acid sequences (DNA and/or RNA) to be amplified. These include the original nucleic acid sequence to be amplified and its complementary second strand as well as either strand of a copy of the original target sequence produced by amplification of the target sequence.
Amplification products, extension products or amplicons are copies of the target sequence or its complementary strand produced by amplification of the target sequence.